Interaction Forces between Colloids and Protein-Coated Surfaces Measured Using an Atomic Force Microscope LI-CHONG XU AND BRUCE E. LOGAN* Department of Civil and Environmental Engineering, The Pennsylvania State University, University Park, Pennsylvania 16802 Bacterial surfaces contain proteins, polysaccharides, and other biopolymers that can affect their adhesion to another surface. To better understand the role of proteins in bacterial adhesion, the interactions between two different model colloids (glass beads and carboxylated latex microspheres) and four proteins covalently bonded to glass surfaces were examined using colloid probes and an atomic force microscope (AFM). Adhesion forces between an uncoated glass colloid probe and protein- coated surfaces, measured in retraction force curves, decreased in the order poly-D-lysine > lysozyme > protein A > BSA. This ordering was consistent with the relative calculated charges of the proteins at neutral pH and the -potentials measured for glass beads and latex microspheres coated with these proteins. When the glass bead was coated with a protein (BSA), overall adhesion forces between the protein-coated colloid and the protein-coated surfaces were reduced, and the adhesion force for each protein decreased in the same order observed in experiments with the uncoated glass bead. When latex colloid probes were coated with BSA, adhesion forces were significantly larger than those measured with BSA-coated glass colloid probes under the same conditions, demonstrating that the nature of the underlying colloid can affect the measured interaction forces. In addition, the adhesion forces measured with the BSA-coated latex colloid increased in a different order (BSA e lysozyme < protein A < poly- D-lysine) than that observed using the BSA-coated glass colloid. It was also found that increasing the solution ionic strength consistently decreased adhesion forces. This result is contrary to the general observation that bacterial adhesion increases with ionic strength. It was speculated that conformational changes of the protein produced this decrease in adhesion with increased ionic strength. These results suggest the need to measure nanoscale adhesion forces in order to understand better molecular scale interactions between colloids and surfaces. Introduction Our understanding of factors that affect the initial attachment of bacteria to a surface is primarily based on colloidal theories that explain adhesion in terms of electrostatic, van der Waals, and acid-base interactions. While extended DLVO (XDLVO) theory has been successful in explaining cell-surface interac- tions under a limited range of conditions (1-4), the theory captures only the general trends in adhesion when a wide range of surfaces and bacteria are examined (5). The primary limitation of the XDLVO theory is that it does not include other types of interactions, such as steric forces due to biopolymers, that are important in bacterial adhesion (6, 7). Solution ionic strength (IS) is well-known to affect microbial adhesion. In general, decreasing the ionic strength decreases bacterial adhesion (3, 5, 8, 9). In packed-bed column tests the stickiness of a particle is calculated using filtration theory on the basis of the particle’s collision efficiency, defined as the probability of attachment based on the predicted number of collisions with the packing material. For example, in minicolumn tests it was found that decreasing the IS from 10 -1 to 10 -5 M decreased the bacterial collision efficiency (R) of Pseudomonas fluorescens P17 by ∼90% (10). This phenomenon is qualitatively consistent with XDLVO theory, as increasing the ionic strength compresses the thickness of the electrostatic double layer and should therefore reduce the repulsion at a given distance between two surfaces. However, the distances above bacteria for which repulsive forces are measured are in general much larger (hundreds of nanometers) than those expected from XDLVO theory (<10 nm) for the solution ionic strengths typically used in experiments (11). XDLVO theory also does not appear to work well at predicting bacterial adhesion when the solution IS exceeds 0.1 M (8). The failure of XDLVO theory to predict microbial adhesion as a function of ionic strength is thought to result from conformational changes of macromolecules, such as proteins and polysaccharides, on the bacterial surface. Biopolymers found on the cell surface include membrane proteins, lipopolysaccharides (LPS), flagella, pillin, and extracellular polymeric substances (EPS) (12). The EPS is a complex mixture of polysaccharides, proteins, nucleic acids, (phos- pho)lipids, and other polymeric compounds (13). Because the composition of EPS is so varied, its effect on bacterial adhesion is not well understood. Abu-Lail and Camesano (14) found that adhesion forces between bacteria and AFM tips increased with ionic strength and concluded that this increase was due to conformational changes in the biopoly- mers on the bacterial cell surface. However, XDLVO theory considers only the charge of such polymers not the steric interactions that these polymers can have with surfaces (either the adhesion surface or the bacterial surface). For microbes lacking EPS, LPS is believed to protrude from the cell into the cell surrounding and therefore to control adhesion (15, 16). For example, the removal of LPS from Escherichia coli JM109 decreased the adhesion force mea- sured using atomic force microscope (AFM) by ∼80% (17). While the length of the LPS can also affect the bacterial adhesion (16), the effect of ionic strength on adhesion was not found to be predictable from macroscopic measurements of bacterial adhesion in packed beds of glass beads (18). Thus, it remains unclear what the role of the LPS on the bacteria surface is relative to other biopolymers, such as proteins, that are on the bacterial surface. The invention and development of AFM has allowed measurement of interaction forces between bacteria and surfaces (tips and colloids) at nano-Newton scales (19, 20). Using the AFM, it is possible to measure physicochemical properties of biopolymers (elasticity, conformation) (21, 22), bacterial cell surface properties such as cell elasticity, and repulsive and attractive interaction forces with bacterial surfaces due to electrostatics and steric interactions (11, 16, 23-28). However, the specific biomolecules interacting with * Corresponding author phone: 814-863-7908; fax: 814-863-7304; e-mail: blogan@psu.edu. Environ. Sci. Technol. 2005, 39, 3592-3600 3592 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 39, NO. 10, 2005 10.1021/es048377i CCC: $30.25  2005 American Chemical Society Published on Web 04/02/2005